The molecular structure of nucleic acids provides for specific detection by means of complementary base pairing of oligonucleotide probes or primers to sequences that are unique to specific target organisms or tissues. Since all biological organisms or specimens contain nucleic acid of specific and defined sequences, a universal strategy for nucleic acid detection has extremely broad applications in a number of diverse research and development areas as well as commercial industries. The potential for practical uses of nucleic acid detection was greatly enhanced by the description of methods to amplify or copy, with fidelity, precise sequences of nucleic acid found at low concentration to much higher copy numbers, so that they are more readily observed by detection methods.
The original nucleic acid amplification method is the polymerase chain reaction (PCR) described by Mullis et al. (U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, and U.S. Pat. No. 4,965,188, all of which are specifically incorporated herein by reference). Subsequent to the introduction of PCR, a wide array of strategies for amplification have been described, such as nucleic acid sequence based amplification (NASBA) (U.S. Pat. No. 5,130,238 to Malek), isothermal methodology (U.S. Pat. No. 5,354,668 to Auerbach), ligase chain reaction (U.S. Pat. No. 5,427,930 to Buirkenmeyer), and strand displacement amplification (SDA), (U.S. Pat. No. 5,455,166 to Walker), all of which are specifically incorporated herein by reference. Some of these amplification strategies, such as SDA or NASBA, require a single stranded nucleic acid target. The target is commonly rendered single stranded via a melting procedure using high temperature prior to amplification.
Prior to nucleic acid amplification and detection, the target nucleic acid must be extracted and purified from the biological specimen such that inhibitors of amplification reaction enzymes are removed. Further, a nucleic acid target that is freely and consistently available for primer annealing must be provided. Numerous strategies for nucleic acid purification are known. These include, for example, phenol-chloroform and/or ethanol precipitation (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)), high salt precipitation (Dykes, Electrophoresis 9:359–368 (1988)), proteinase K digestion (Grimberg et al., Nucleic Acids Res., 22:8390 (1989)), chelex and other boiling methods (Walsh et al., Bio/techniques 10:506–513 (1991)) and solid phase binding and elution (Vogelstein and Gillespie, Proc. Nat. Acad. Sci. USA, 76:615–619 (1979)), all of which are specifically incorporated herein by reference.
The analysis of nucleic acid targets therefore consists of three steps: nucleic acid extraction/purification from biological specimens, direct probe hybridization and/or amplification of the specific target sequence, and specific detection thereof. In conventional protocols each of these three steps is performed separately, thus making nucleic acid analysis labor intensive. Further, numerous manipulations, instruments and reagents are necessary to perform each step of the analysis. Another concern with current methodologies is the significant chance of specimen cross-contamination, for example, between concurrently run specimens or from a previously amplified sample.
For analysis purposes, nucleic acid must frequently be extracted from extremely small specimens from which it is difficult, if not impossible, to obtain a second confirmatory specimen. Examples include analysis of crime scene evidence or fine needle biopsies for clinical testing. In such examples, the extent of the genetic testing and confirmation through replica testing is thus limited by the nucleic acid specimen size. Using conventional extraction protocols for these small specimens, the nucleic acid is often lost or yields are such that only a single or few amplification analyses are possible.
The requirements for binding of DNA to solid phases and subsequently being able to elute the DNA therefrom have been described by Boom (U.S. Pat. No. 5,234,809, which is specifically incorporated herein by reference) and Woodard (U.S. Pat. No. 5,405,951, U.S. Pat. No. 5,438,129, U.S. Pat. No. 5,438,127, all of which are specifically incorporated herein by reference). Specifically, DNA binds to solid phases that are electropositive and hydrophilic. Electropositive elements can be rendered sufficiently hydrophilic by hydroxyl (—OH) or other groups, resulting in a solid phase matrix that tightly binds DNA, while proteins or inhibitors do not bind to the solid phase matrix. Since conventional purification methods require elution of the bound nucleic acid, solid phase matrices that bind nucleic acids but do not allow substantially complete elution have been described as being of no use for DNA purification. In fact, considerable effort has been expended to derive solid phase matrices sufficiently hydrophilic to adequately bind nucleic acid and yet allow for its elution therefrom (See, for example, U.S. Pat. Nos. 5,523,392, 5,525,319 and 5,503,816, all to Woodard and all of which are specifically incorporated herein by reference).
Boom, supra, describes solid phase DNA amplification using high chaotropic salt to reversibly bind the DNA to silica. However, when the silica-bound DNA is placed in the amplification reaction buffer, the nucleic acid is actually eluted from the silica. Therefore, the amplification according to the method of Boom actually occurs in solution, not on solid phase. Furthermore, since the nucleic acid is eluted from the solid phase prior to amplification, the amplification can only be performed once.
Del Rio et al., Bio/techniques 20:970–974 (1996)) describe filter entrapment of nucleic acid in a manner allowing for repeat amplification. However, they do not describe a binding mechanism that is irreversible, and therefore the method is only recommended for analysis of higher nucleic acid concentrations, and then only for a limited number of analyses.
It would be advantageous to directly integrate nucleic acid purification and/or extraction with other nucleic acid analyses and/or manipulations so as to simplify the analysis procedure and methodologies, as well as reduce and/or remove the risk of cross-contamination. It further would be advantageous to eliminate the melt step necessary for generating single strand nucleic acid for probe hybridization or amplification primer annealing.